Ad P. IJzerman

1,3-dialkylxanthine derivatives having high potency as antagonists at human A2B adenosine receptors

The structure–activity relationships (SAR) of alkylxanthine derivatives as antagonists at the recombinant human adenosine receptors were explored in order to identify selective antagonists of A2B receptors. The effects of lengthening alkyl substituents from methyl to butyl at 1‐ and 3‐positions and additional substitution at the 7‐ and 8‐positions were probed. Ki values, determined in competition binding in membranes of HEK‐293 cells expressing A2B receptors using 125I‐ABOPX (125I‐3‐(4‐amino‐3‐iodobenzyl)‐8‐(phenyl‐4‐oxyacetate)‐1‐propylxanthine), were approximately 10 to 100 nM for 8‐phenylxanthine functionalized congeners. Xanthines containing 8‐aryl, 8‐alkyl, and 8‐cycloalkyl substituents, derivatives of XCC (8‐[4‐[[[carboxy]methyl]oxy]phenyl]‐1,3‐dipropylxanthine) and XAC (8‐[4‐[[[[(2‐aminoethyl)amino]carbonyl]methyl]‐oxy]phenyl]‐1,3‐dipropylxanthine), containing various ester and amide groups, including L‐ and D‐amino acid conjugates, were included. Enprofylline was 2‐fold more potent than theophylline in A2B receptor binding, and the 2‐thio modification was not tolerated. Among the most potent derivatives examined were XCC, its hydrazide and aminoethyl and fluoroethyl amide derivatives, XAC, N‐hydroxyethyl‐XAC, and the L‐citrulline and D‐p‐aminophenylalanine conjugates of XAC. An N‐hydroxysuccinimide ester of XCC (XCC‐NHS, MRS 1204) bound to A2B receptors with a Ki of 9.75 nM and was the most selective (at least 20‐fold) in this series. In a functional assay of recombinant human A2B receptors, four of these potent xanthines were shown to fully antagonize the effects of NECA‐induced stimulation of cyclic AMP accumulation. Drug Dev. Res. 47:45–53, 1999. Published 1999 Wiley‐Liss, Inc. This article is a US Government work and, as such, is in the public domain in the United States of America.

Potent antagonists for the human adenosine A2B receptor. Derivatives of the triazolotriazine adenosine receptor antagonist ZM241385 with high affinity

A series of novel and known 5‐substituted 7‐amino‐2‐(2‐furyl)[1,2,4]triazolo[1,5‐a][1,3,5]triazine derivatives were synthesized and tested for adenosine receptor antagonism in radioligand binding assays at all four adenosine receptor subtypes and for inhibition of the agonist‐induced cyclic AMP response at human A2B receptors. The known potent adenosine A2A receptor antagonist, 7‐amino‐2‐(2‐furyl)‐5‐[2‐(4‐hydroxyphenyl)ethyl]amino[1,2,4]triazolo[1,5‐a][1,3,5]triazine (ZM241385, KiA2A = 1.78 nM) had a Ki value of 16.5 nM at A2B receptors in radioligand binding studies on Chinese hamster ovary cells expressing A2B receptors. A pA2 value of 7.9 was measured for the inhibition of the cyclic AMP response by A2B receptors induced by 5′‐N‐ethylcarboxamidoadenosine (NECA). In a series of 5‐phenyl(alkyl)amino analogs the 5‐(2‐phenylethyl)amino analog LUF5452 and the 5‐benzylamino analog LUF5451 were both more potent than ZM241385 in the cyclic AMP assay at A2B receptors. Moreover, Ki values of 9.9 and 7.6 nM were found in binding studies at this receptor subtype, indicating that LUF5451 and LUF5452 are more potent A2B receptor antagonists than ZM241385. The affinity of LUF5451 for the A2A receptor (Ki value = 13 nM) showed that the selectivity for this receptor subtype was lost and that a modest A2B receptor selectivity was achieved. The 5‐(2‐phenylhydrazino) derivative LUF5475 showed a high A2B receptor affinity (Ki = 7.6 nM), while it was equally active at A2A receptors, being A2B receptor‐selective with respect to A1 and A3 receptors. Drug Dev. Res. 48:95–103, 1999.

Molecular modeling of adenosine receptors. The ligand binding site on the rat adenosine A2A receptor

The amino acid sequence of the rat adenosine A2A receptor and the atomic coordinates of bacteriorhodopsin were combined to generate a three-dimensional model for the adenosine A2A receptor. This model consists of seven amphipathic alpha-helices, forming a pore that is rather hydrophilic compared to the hydrophobic outside of the protein. Subsequently, a highly potent and selective ligand for this receptor, 2-(cyclohexylmethylidinehydrazino)adenosine (SHA 174), was docked into this cavity. A binding site is proposed that takes into account the conformational characteristics of the ligand. Moreover, it involves two histidine residues that were shown to be important for ligand coordination from chemical modification studies. Subsequently, the deduced binding site was used to model other potent ligands, including 8-(3-chlorostyryl)caffeine, a new A2-selective antagonist, that could all be accommodated consistent with earlier biochemical and pharmacological findings. Finally, some thoughts on how adenosine receptor activation might proceed are put forward, based on structural analogies with the enzyme family of serine proteases.

Functional selectivity of adenosine receptor ligands

Adenosine receptors are plasma membrane proteins that transduce an extracellular signal into the interior of the cell. Basically every mammalian cell expresses at least one of the four adenosine receptor subtypes. Recent insight in signal transduction cascades teaches us that the current classification of receptor ligands into agonists, antagonists, and inverse agonists relies very much on the experimental setup that was used. Upon activation of the receptors by the ubiquitous endogenous ligand adenosine they engage classical G protein-mediated pathways, resulting in production of second messengers and activation of kinases. Besides this well-described G protein-mediated signaling pathway, adenosine receptors activate scaffold proteins such as β-arrestins. Using innovative and sensitive experimental tools, it has been possible to detect ligands that preferentially stimulate the β-arrestin pathway over the G protein-mediated signal transduction route, or vice versa. This phenomenon is referred to as functional selectivity or biased signaling and implies that an antagonist for one pathway may be a full agonist for the other signaling route. Functional selectivity makes it necessary to redefine the functional properties of currently used adenosine receptor ligands and opens possibilities for new and more selective ligands. This review focuses on the current knowledge of functionally selective adenosine receptor ligands and on G protein-independent signaling of adenosine receptors through scaffold proteins.

Interaction of amiloride and its analogues with adenosine A1 receptors in calf brain

Amiloride, a potassium sparing diuretic, is known to interact with a number of ion transport systems, receptors and enzymes. Here, we report on the interaction between this drug and the adenosine A1 receptor as present in calf brain membranes. Adenosine A1 receptors are characterized by a subnanomolar affinity for the antagonists [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX) and the agonist [3H]N6-R-1-phenyl-2-propyladenosine ([3H]PIA). Amiloride displaces both agonist and antagonist binding with a Ki value in the low micromolar range. This inhibition is counteracted by NaCl and protons, in contrast to the binding of [3H]PIA and [3H]DPCPX. The results suggest that amiloride interacts with the adenosine A1 receptor at a site distinct from the ligand binding site. In order to elucidate the role of one of the ion transport systems known to be inhibited by amiloride, eight amiloride analogues with different sensitivities for these systems were tested. The potency and order of potency of these compounds towards adenosine A1 receptors excludes the involvement of the epithelial Na+ channel, Na+/H+ exchanger or Na+/Ca2+ exchanger.

Elucidation of structure-activity relationships of 2-amino-3-benzoylthiophenes : Study of their allosteric enhancing vs. antagonistic activity on adenosine A1 receptors

Novel 2‐amino‐3‐benzoylthiophene derivatives, with variable substitution on the thiophene as well as benzoyl ring, were synthesized and evaluated both as allosteric enhancers of agonist binding to the rat adenosine A1 receptor, and as antagonists on this receptor. Structural features were identified on the novel derivatives that favored allosteric enhancing activity, such as benzoyl lipophilic substitution and thiophene 4‐alkyl substitution. In contrast, antagonistic properties were favored by thiophene 5‐bulky substitution. Upon further analysis, a significant correlation was found between antagonistic activity and hydrophobic fragment constants (π values) of substituent R5, in contrast to a negative correlation with those of R4. Comparison of low energy conformations of some of the 2‐amino‐3‐benzoylthiophene derivatives (PD81,723 and 4f) with known adenosine A1 antagonists (theophylline and 8‐cyclohexyltheophylline) indicated that thiophene 5‐substituents may interact with the same lipophilic domain of the adenosine A1 receptor accommodating 8‐substituents of xanthine antagonists. Drug Dev. Res. 49:227–237, 2000.

Biological and Pharmacological Roles of HCA Receptors

The hydroxy-carboxylic acid (HCA) receptors HCA(1), HCA(2), and HCA(3) were previously known as GPR81, GPR109A, and GPR109B, respectively, or as the nicotinic acid receptor family. They form a cluster of G protein-coupled receptors with high sequence homology. Recently, intermediates of energy metabolism, all HCAs, have been reported as endogenous ligands for each of these receptors. The HCA receptors are predominantly expressed on adipocytes and mediate the inhibition of lipolysis by coupling to G(i)-type proteins. HCA(1) is activated by lactate, HCA(2) by the ketone body 3-hydroxy-butyrate, and HCA(3) by hydroxylated β-oxidation intermediates, especially 3-hydroxy-octanoic acid. Both HCA(2) and HCA(3) are part of a negative feedback loop which keeps the release of fat stores in check under starvation conditions, whereas HCA(1) plays a role in the antilipolytic (fat-conserving) effect of insulin. HCA(2) was first discovered as the molecular target of the antidyslipidemic drug nicotinic acid (or niacin). Many synthetic agonists have since been designed for HCA(2) and HCA(3), but the development of a new, improved HCA-targeted drug has not been successful so far, despite a number of clinical studies. Recently, it has been shown that the major side effect of nicotinic acid, skin flushing, is mediated by HCA(2) receptors on keratinocytes, as well as on Langerhans cells in the skin. In this chapter, we summarize the latest developments in the field of HCA receptor research, with emphasis on (patho)physiology, receptor pharmacology, major ligand classes, and the therapeutic potential of HCA ligands.

Allosteric modulation of adenosine receptors

Allosteric modulators for adenosine receptors may have potential therapeutic advantage over orthosteric ligands. Allosteric enhancers at the adenosine A1 receptor have been linked to antiarrhythmic and antilipolytic activity. They may also have therapeutic potential as analgesics and neuroprotective agents. A3 allosteric enhancers are postulated to be useful against ischemic conditions or as antitumor agents. In this review, we address recent developments regarding the medicinal chemistry of such compounds. Most efforts have been and are directed toward adenosine A1 and A3 receptors, whereas limited or no information is available for A2A and A2B receptors. We also discuss some findings, mostly receptor mutation studies, regarding localization of the allosteric binding sites on the receptors.

Partial agonism of theophylline-7-riboside on adenosine receptors

Theophylline-7-riboside was evaluated as a partial agonist for rat adenosine receptors. Radioligand binding experiments were performed on both A1 and A2a adenosine receptors, using several methodologies to discriminate between agonists and antagonists. Mainly from thermodynamic data it was concluded that on A1 receptors theophylline-7-riboside had characteristics intermediate between full agonists, such as N6-cyclopentyladen-osine, and full antagonists, such as the xanthines. The partial agonistic behaviour of theophylline-7-riboside was further explored in second messenger studies in intact cells. In FRTL-5 rat thyroid cells theophylline-7-riboside behaved as a partial agonist for A1 receptors, slightly inhibiting forskolin-stimulated cyclic AMP levels.The implications of these biochemical findings were further analysed in in vivo pharmacology. The infusion of theophylline-7-riboside in conscious, normotensive rats led to marked changes in cardiovascular parameters, although less outspoken than observed with full agonists for either A1 or A2a receptors. The concomitant determination of the blood concentrations of theophylline-7-riboside and its metabolite theophylline allowed the estimation of in vivo pharmacokinetic and pharmacodynamic parameters. Thus, the EC50 value of theophylline-7-riboside for lowering the mean arterial pressure was 47±12 μg/ml blood. The short duration of action of theophylline-7-riboside makes it improbable that its metabolite theophylline interferes with its effects.In conclusion, theophylline-7-riboside is one of the first partial agonists for adenosine receptors. It may serve as a tool in further investigations of adenosine receptor partial agonism.

The adhesion G protein-coupled receptor G2 (ADGRG2/GPR64) constitutively activates SRE and NFκB and is involved in cell adhesion and migration

Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which originally was described to be expressed in the epididymis and studied for its potential role in male fertility, is highly up-regulated in a number of carcinomas, including breast cancer. Here, we demonstrate that ADGRG2 is a functional receptor, which in transfected HEK293 cells signals with constitutive activity through the adhesion- and migration-related transcription factors serum response element (SRE) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) presumably via coupling to Gα12/13 and Gαq. However, activation of these two pathways appears to occur through distinct molecular activation mechanisms as auto-proteolytic cleavage is essential for SRE activation but not required for NFκB signaling. The overall activation mechanism for ADGRG2 is clearly distinct from the established ADGR activation mechanism as it requires the large extracellular N-terminal domain for proper intracellular signal transduction. Knockdown of ADGRG2 by siRNA in the highly motile breast cancer cell lines Hs578T and MDA-MB-231 resulted in a strong reduction in cell adhesion and subsequent cell migration which was associated with a selective reduction in RelB, an NFκB family member. It is concluded that the adhesion GPCR ADGRG2 is critically involved in the adhesion and migration of certain breast cancer cells through mechanisms including a non-canonical NFkB pathway and that ADGRG2 could be a target for treatment of certain types of cancer.